Significance The PH-20 protein is one of several molecules on the surface of mammalian sperm which interact with the oocyte investments prior to fertilization. PH-20 appears to have multiple functions and is a target for contraceptive vaccines that are now being developed and tested for effectiveness in primates. Objectives In this project we are studying the cellular mechanisms of action of PH-20 during primate fertilization because it is applicable to mammalian fertilization biology and will be particularly useful in the design of contraceptive vaccines with multi determinant immunogens. Results PH-20 is a hyaluronidase and a requirement for PH-20 function during sperm penetration of the cumulus extra cellular matrix (ECM) is highly likely. Two primary isoforms of macaque sperm PH -20 can be distinguished by their molecular weights of 64 kDa and 53 kDa. Both of these forms of PH-20 are found in three separate locations or compartments of the sperm. Treatment of sperm with hyaluronic acid (HA) results in phosphorylation of sperm proteins, and an increase in intracellular calcium. These HA-induced changes can be inhibited by treatment with Fab fragments of anti-PH-20 IgG. We can show increases in sperm calcium as measured in individual cells during penetration of the macaque cumulus ECM in vitro. The role of HA in mediating such intracellular calcium changes is suggested by similar observations on individual sperm in artificial gels containing HA. The biological importance of intracellular calcium changes may be to [unreadable]prime[unreadable] sperm for immediate induction of the acrosome reaction as soon as sperm binding to the zona pellucida takes place. PH-20 may have a required function in fertilization, making it a vulnerable target for contraceptive agents. Future Directions Determine structures of macaque PH-20 isoforms, characterize hyaluronidase and HA binding activities and investigate signaling functions during interaction with the cumulus ECM and zona pellucida. KEYWORDS sperm, fertilization, contraception